The goal of the proposed research is to develop and microscale separation methods of both high resolving power and high sensitivity. Microcolumn liquid chromatography has demonstrated its potential for providing great resolving power for the analysis of complex mixtures. We are now investigating the potential of multiplying this resolving power by developing systems for comprehensive two-dimensional liquid chromatography. This extraordinary separating power creates the potential for analysis of mixtures containing thousands of detectable components. When combined with a sensitive detection technique such as laser induced fluorescence (LIF), a micro LC-LC-LIF system might be capable of determining the levels of dozens of bioactive peptides in one run from a single drop of blood. Sensitive detection must be expanded beyond those compounds which can easily be labeled with a fluorescent tag or are inherently electroactive (easily oxidized or reduced). The electrospray process provides a remarkable means for introducing non-volatile substances into the gas phase or vacuum. We plan to make use of the electrospray process as a means to introduce non-volatile species into the gas phase for detection by the gas phase particle counting method of condensation nucleation counting. This technique permits counting of protein molecules of molecular weight above 10,000 daltons. We also plan to investigate electrospray as a means to introduce non-volatile species into a photoionization detector. We will continue our investigations of electrospray mass spectrometry. We have developed considerable technical capability for handling and analyzing the contents of single cells. We are expanding that capability, by developing an instrument capable of sizing and analyzing the contents of single vesicles. We plan to continue these studies, and bring the new developments in two-dimensional separation systems and sensitive detection schemes to bear on these problems. In this way we hope to measure substances at 1,000-fold lower levels, such as bioactive peptides, in single cells.